Symbiotic nitrogen fixation does not stimulate soil phosphatase activity under temperate and tropical trees.

Symbiotic nitrogen (N)-fixing plants can enrich ecosystems with N, which can alter the cycling and demand for other nutrients. Researchers have hypothesized that fixed N could be used by plants and soil microbes to produce extracellular phosphatase enzymes, which release P from organic matter. Consistent with this speculation, the presence of N-fixing plants is often associated with high phosphatase activity, either in the soil or on root surfaces, although other studies have not found this association, and the connection between phosphatase and rates of N fixation-the mechanistic part of the argument-is tenuous. Here, we measured soil phosphatase activity under N-fixing trees and non-fixing trees transplanted and grown in tropical and temperate sites in the USA: two sites in Hawaii, and one each in New York and Oregon. This provides a rare example of phosphatase activity measured in a multi-site field experiment with rigorously quantified rates of N fixation. We found no difference in soil phosphatase activity under N-fixing vs. non-fixing trees nor across rates of N fixation, though we note that no sites were P limited and only one was N limited. Our results add to the literature showing no connection between N fixation rates and phosphatase activity.

Divergent Pathways of Nitrogen-Fixing Trees through Succession Depend on Starting Nitrogen Supply and Priority Effects.

AbstractNitrogen-fixing trees are a major potential source of nitrogen in terrestrial ecosystems. The degree to which they persist in older forests has considerable implications for forest nitrogen budgets. We characterized nitrogen-fixing tree abundance across stand age in the contiguous United States and analyzed a theoretical model to help understand competitive outcomes and successional trajectories of nitrogen-fixing and nonfixing trees. Nitrogen-fixing tree abundance is bimodal in all regions except the northeastern United States, even in older forests, suggesting that competitive exclusion (including priority effects) is more common than coexistence at the spatial scale of our analysis. Our model analysis suggests conditions under which alternative competitive outcomes are possible and when they are transient (lasting decades or centuries) versus persistent (millennia). Critically, the timescale of the feedbacks between nitrogen fixation and soil nitrogen supply, which is thought to drive the exclusion of nitrogen-fixing trees through succession, can be long. Therefore, the long transient outcomes of competition are more relevant for real forests than the long-term equilibrium. Within these long-term transients, the background soil nitrogen supply is a major determinant of competitive outcomes. Consistent with the expectations of resource ratio theory, competitive exclusion is more likely at high and low nitrogen supply, while intermediate nitrogen supply makes coexistence or priority effects possible. However, these outcomes are modified by the nitrogen fixation strategy: obligate nitrogen fixation makes coexistence more likely than priority effects, compared with perfectly facultative fixation. These results advance our understanding of the successional trajectories of nitrogen-fixing trees and their effects on ecosystem development in secondary succession.

Light, nitrogen supply, and neighboring plants dictate costs and benefits of nitrogen fixation for seedlings of a tropical nitrogen-fixing tree.

The ability to fix nitrogen may confer a competitive advantage or disadvantage to symbiotic nitrogen-fixing plants depending on the availability of soil nitrogen and energy to fuel fixation. Understanding these costs and benefits of nitrogen fixation is critical to predicting ecosystem dynamics and nutrient cycling. We grew inoculated (with symbiotic bacteria) and uninoculated seedlings of Pentaclethra macroloba (a nitrogen-fixing tree species) both in isolation and with Virola koschnyi (a nonfixing species) under gradients of light and soil nitrogen to assess how the ability to fix nitrogen and fixation activity affect growth, biomass allocation, and responses to neighboring plants. Inoculation itself did not provide a growth advantage to nitrogen fixers, regardless of nitrogen limitation status. Higher nitrogen fixation rates increased biomass growth similarly for nitrogen-limited and nitrogen-saturated fixers. Nodule production was offset by reduced fine-root biomass for inoculated nitrogen fixers, resulting in no change in total belowground allocation associated with nitrogen fixation. Under nitrogen-limited conditions, inoculated nitrogen fixers partially downregulated fixation in the presence of a nonfixing neighbor. These results suggest that nitrogen fixation can provide a growth advantage, even under nitrogen-saturated conditions, and that nitrogen fixers may reduce fixation rates to minimize facilitation of neighbors.

Effects of two centuries of global environmental variation on phenology and physiology of Arabidopsis thaliana.

Intraspecific trait variation is caused by genetic and plastic responses to environment. This intraspecific diversity is captured in immense natural history collections, giving us a window into trait variation across continents and through centuries of environmental shifts. Here we tested if hypotheses based on life history and the leaf economics spectrum explain intraspecific trait changes across global spatiotemporal environmental gradients. We measured phenotypes on a 216-year time series of Arabidopsis thaliana accessions from across its native range and applied spatially varying coefficient models to quantify region-specific trends in trait coordination and trait responses to climate gradients. All traits exhibited significant change across space or through time. For example, δ15 N decreased over time across much of the range and leaf C:N increased, consistent with predictions based on anthropogenic changes in land use and atmosphere. Plants were collected later in the growing season in more recent years in many regions, possibly because populations shifted toward more spring germination and summer flowering as opposed to fall germination and spring flowering. When climate variables were considered, collection dates were earlier in warmer years, while summer rainfall had opposing associations with collection date depending on regions. There was only a modest correlation among traits, indicating a lack of a single life history/physiology axis. Nevertheless, leaf C:N was low for summer- versus spring-collected plants, consistent with a life history-physiology axis from slow-growing winter annuals to fast-growing spring/summer annuals. Regional heterogeneity in phenotype trends indicates complex responses to spatiotemporal environmental gradients potentially due to geographic genetic variation and climate interactions with other aspects of environment. Our study demonstrates how natural history collections can be used to broadly characterize trait responses to environment, revealing heterogeneity in response to anthropogenic change.

Meta-analysis on the potential for increasing nitrogen losses from intensifying tropical agriculture.

Fertilized temperate croplands export large amounts of reactive nitrogen (N), which degrades water and air quality and contributes to climate change. Fertilizer use is poised to increase in the tropics, where widespread food insecurity persists and increased agricultural productivity will be needed, but much less is known about the potential consequences of increased tropical N fertilizer application. We conducted a meta-analysis of tropical field studies of nitrate leaching, nitrous oxide emissions, nitric oxide emissions, and ammonia volatilization totaling more than 1,000 observations. We found that the relationship between N inputs and losses differed little between temperate and tropical croplands, although total nitric oxide losses were higher in the tropics. Among the potential drivers we studied, the N input rate controlled all N losses, but soil texture and water inputs also controlled hydrological N losses. Irrigated systems had significantly higher losses of ammonia, and pasture agroecosystems had higher nitric oxide losses. Tripling of fertilizer N inputs to tropical croplands from 50 to 150 kg N ha-1  year-1 would have substantial environmental implications and would lead to increases in nitrate leaching (+30%), nitrous oxide emissions (+30%), nitric oxide (+66%) emissions, and ammonia volatilization (+74%), bringing tropical agricultural nitrate, nitrous oxide, and ammonia losses in line with temperate losses and raising nitric oxide losses above them.

A mechanism of expansion: Arctic deciduous shrubs capitalize on warming-induced nutrient availability.

Warming-induced nutrient enrichment in the Arctic may lead to shifts in leaf-level physiological properties and processes with potential consequences for plant community dynamics and ecosystem function. To explore the physiological responses of Arctic tundra vegetation to increasing nutrient availability, we examined how a set of leaf nutrient and physiological characteristics of eight plant species (representing four plant functional groups) respond to a gradient of experimental nitrogen (N) and phosphorus (P) enrichment. Specifically, we examined a set of chlorophyll fluorescence measures related to photosynthetic efficiency, performance and stress, and two leaf nutrient traits (leaf %C and %N), across an experimental nutrient gradient at the Arctic Long Term Ecological Research site, located in the northern foothills of the Brooks Range, Alaska. In addition, we explicitly assessed the direct relationships between chlorophyll fluorescence and leaf %N. We found significant differences in physiological and nutrient traits between species and plant functional groups, and we found that species within one functional group (deciduous shrubs) have significantly greater leaf %N at high levels of nutrient addition. In addition, we found positive, saturating relationships between leaf %N and chlorophyll fluorescence measures across all species. Our results highlight species-specific differences in leaf nutrient traits and physiology in this ecosystem. In particular, the effects of a gradient of nutrient enrichment were most prominent in deciduous plant species, the plant functional group known to be increasing in relative abundance with warming in this ecosystem.

Nitrogen-fixing trees inhibit growth of regenerating Costa Rican rainforests.

More than half of the world's tropical forests are currently recovering from human land use, and this regenerating biomass now represents the largest carbon (C)-capturing potential on Earth. How quickly these forests regenerate is now a central concern for both conservation and global climate-modeling efforts. Symbiotic nitrogen-fixing trees are thought to provide much of the nitrogen (N) required to fuel tropical secondary regrowth and therefore to drive the rate of forest regeneration, yet we have a poor understanding of how these N fixers influence the trees around them. Do they promote forest growth, as expected if the new N they fix facilitates neighboring trees? Or do they suppress growth, as expected if competitive inhibition of their neighbors is strong? Using 17 consecutive years of data from tropical rainforest plots in Costa Rica that range from 10 y since abandonment to old-growth forest, we assessed how N fixers influenced the growth of forest stands and the demographic rates of neighboring trees. Surprisingly, we found no evidence that N fixers facilitate biomass regeneration in these forests. At the hectare scale, plots with more N-fixing trees grew slower. At the individual scale, N fixers inhibited their neighbors even more strongly than did nonfixing trees. These results provide strong evidence that N-fixing trees do not always serve the facilitative role to neighboring trees during tropical forest regeneration that is expected given their N inputs into these systems.

Nitrogen-fixing tree abundance in higher-latitude North America is not constrained by diversity.

The rarity of nitrogen (N)-fixing trees in frequently N-limited higher-latitude (here, > 35°) forests is a central biogeochemical paradox. One hypothesis for their rarity is that evolutionary constraints limit N-fixing tree diversity, preventing N-fixing species from filling available niches in higher-latitude forests. Here, we test this hypothesis using data from the USA and Mexico. N-fixing trees comprise only a slightly smaller fraction of taxa at higher vs. lower latitudes (8% vs. 11% of genera), despite 11-fold lower abundance (1.2% vs. 12.7% of basal area). Furthermore, N-fixing trees are abundant but belong to few species on tropical islands, suggesting that low absolute diversity does not limit their abundance. Rhizobial taxa dominate N-fixing tree richness at lower latitudes, whereas actinorhizal species do at higher latitudes. Our results suggest that low diversity does not explain N-fixing trees' rarity in higher-latitude forests. Therefore, N limitation in higher-latitude forests likely results from ecological constraints on N fixation.

Feedbacks between plant N demand and rhizosphere priming depend on type of mycorrhizal association.

Ecosystem carbon (C) balance is hypothesised to be sensitive to the mycorrhizal strategies that plants use to acquire nutrients. To test this idea, we coupled an optimality-based plant nitrogen (N) acquisition model with a microbe-focused soil organic matter (SOM) model. The model accurately predicted rhizosphere processes and C-N dynamics across a gradient of stands varying in their relative abundance of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) trees. When mycorrhizal dominance was switched - ECM trees dominating plots previously occupied by AM trees, and vice versa - legacy effects were apparent, with consequences for both C and N stocks in soil. Under elevated productivity, ECM trees enhanced decomposition more than AM trees via microbial priming of unprotected SOM. Collectively, our results show that ecosystem responses to global change may hinge on the balance between rhizosphere priming and SOM protection, and highlight the importance of dynamically linking plants and microbes in terrestrial biosphere models.

The symbionts made me do it: legumes are not hardwired for high nitrogen concentrations but incorporate more nitrogen when inoculated.

High tissue nitrogen (N) concentrations in N-fixing legumes may be driven by an evolutionary commitment to a high N strategy, by higher N availability from fixation, or by some other cause. To disentangle these hypotheses, we asked two questions: are legumes hardwired to have high N concentrations? Aside from delivering fixed N, how does inoculation affect legume N concentrations? In order to understand drivers of plant stoichiometry, we subjected four herbaceous legume species to nine levels of N fertilization in a glasshouse. Half of the individuals were inoculated with crushed nodules, whereas the other half remained uninoculated and could not fix N. Across four legume species, we found that tissue stoichiometry and nutrient content were more plastic than has been described for any other plant species. In addition, inoculated plants had higher tissue N concentrations than N fixation activity alone can explain. Rather than being hardwired for high N or phosphorus (P) demand, the legumes we examined were highly flexible in their nutrient allocation. Understanding the drivers of legume N concentrations is essential to understanding the role of N fixers in community- and ecosystem-level processes.

Spatial heterogeneity can resolve the nitrogen paradox of tropical forests.

Many tropical forests are characterized by large losses of plant-available forms of nitrogen (N), indicating that they are N rich, and by an abundance of plants capable of symbiotic N fixation. These N-fixing plants can fix enough N to drive N richness. However, biological N fixation (BNF) is more expensive than using plant-available N, so sustained BNF in N-rich soils appears to be a paradox. Here, we use spatially explicit ecosystem models to analyze the conditions under which spatial heterogeneity can induce simultaneous BNF and loss of plant-available N (hereafter, we call this combination "N-rich BNF"). Spatial movement of litter to neighboring plants' rooting zones can maintain N-rich BNF under a variety of conditions. For example, when N-fixers have higher N demand than non-fixers, N-fixers export N-rich litter to non-fixers, inducing large losses of plant-available N from the ecosystem, and receive N-poor litter from non-fixers, inducing BNF. BNF and N loss fluxes increase in proportion to the ratio of N-fixer litter N:P to non-fixer litter N:P, and also in proportion to the fraction of litter transferred out of a tree's rooting zone. Stoichiometric variability augments N-rich BNF, as does increasing the fraction of the landscape occupied by N-fixers, at least when they are rare. On the contrary, greater root overlap between neighbors and clumping of N-fixers diminish N-rich BNF. Finally, we examined how spatial litter transfer interacts with another mechanism that can sustain N-rich BNF, incomplete down-regulation of BNF. Spatial transfer and incomplete down-regulation can both sustain N-rich BNF, but they are compensatory rather than additive. These mechanisms can be distinguished by examining where N losses occur. Incomplete down-regulation of BNF leads to greater N loss under N-fixing trees, whereas spatial litter transfer leads to greater N loss under non-fixing trees. Along with time lags in regulating BNF, these results comprise a series of hypotheses that could help understand the N paradox of tropical forests.

Why are nitrogen-fixing trees rare at higher compared to lower latitudes?

Symbiotic nitrogen (N) fixation provides a dominant source of new N to the terrestrial biosphere, yet in many cases the abundance of N-fixing trees appears paradoxical. N-fixing trees, which should be favored when N is limiting, are rare in higher latitude forests where N limitation is common, but are abundant in many lower latitude forests where N limitation is rare. Here, we develop a graphical and mathematical model to resolve the paradox. We use the model to demonstrate that N fixation is not necessarily cost effective under all degrees of N limitation, as intuition suggests. Rather, N fixation is only cost effective when N limitation is sufficiently severe. This general finding, specific versions of which have also emerged from other models, would explain sustained moderate N limitation because N-fixing trees would either turn N fixation off or be outcompeted under moderate N limitation. From this finding, four general hypothesis classes emerge to resolve the apparent paradox of N limitation and N-fixing tree abundance across latitude. The first hypothesis is that N limitation is less common at higher latitudes. This hypothesis contradicts prevailing evidence, so is unlikely, but the following three hypotheses all seem likely. The second hypothesis, which is new, is that even if N limitation is more common at higher latitudes, more severe N limitation might be more common at lower latitudes because of the capacity for higher N demand. Third, N fixation might be cost effective under milder N limitation at lower latitudes but only under more severe N limitation at higher latitudes. This third hypothesis class generalizes previous hypotheses and suggests new specific hypotheses. For example, greater trade-offs between N fixation and N use efficiency, soil N uptake, or plant turnover at higher compared to lower latitudes would make N fixation cost effective only under more severe N limitation at higher latitudes. Fourth, N-fixing trees might adjust N fixation more at lower than at higher latitudes. This framework provides new hypotheses to explain the latitudinal abundance distribution of N-fixing trees, and also provides a new way to visualize them. Therefore, it can help explain the seemingly paradoxical persistence of N limitation in many higher latitude forests.

Global climate change will increase the abundance of symbiotic nitrogen-fixing trees in much of North America.

Symbiotic nitrogen (N)-fixing trees can drive N and carbon cycling and thus are critical components of future climate projections. Despite detailed understanding of how climate influences N-fixation enzyme activity and physiology, comparatively little is known about how climate influences N-fixing tree abundance. Here, we used forest inventory data from the USA and Mexico (>125,000 plots) along with climate data to address two questions: (1) How does the abundance distribution of N-fixing trees (rhizobial, actinorhizal, and both types together) vary with mean annual temperature (MAT) and precipitation (MAP)? (2) How will changing climate shift the abundance distribution of N-fixing trees? We found that rhizobial N-fixing trees were nearly absent below 15°C MAT, but above 15°C MAT, they increased in abundance as temperature rose. We found no evidence for a hump-shaped response to temperature throughout the range of our data. Rhizobial trees were more abundant in dry than in wet ecosystems. By contrast, actinorhizal trees peaked in abundance at 5-10°C MAT and were least abundant in areas with intermediate precipitation. Next, we used a climate-envelope approach to project how N-fixing tree relative abundance might change in the future. The climate-envelope projection showed that rhizobial N-fixing trees will likely become more abundant in many areas by 2080, particularly in the southern USA and western Mexico, due primarily to rising temperatures. Projections for actinorhizal N-fixing trees were more nuanced due to their nonmonotonic dependence on temperature and precipitation. Overall, the dominant trend is that warming will increase N-fixing tree abundance in much of the USA and Mexico, with large increases up to 40° North latitude. The quantitative link we provide between climate and N-fixing tree abundance can help improve the representation of symbiotic N fixation in Earth System Models.

Higher survival drives the success of nitrogen-fixing trees through succession in Costa Rican rainforests.

Trees capable of symbiotic nitrogen (N) fixation ('N fixers') are abundant in many tropical forests. In temperate forests, it is well known that N fixers specialize in early-successional niches, but in tropical forests, successional trends of N-fixing species are poorly understood. We used a long-term census study (1997-2013) of regenerating lowland wet tropical forests in Costa Rica to document successional patterns of N fixers vs non-fixers, and used an individual-based model to determine the demographic drivers of these trends. N fixers increased in relative basal area during succession. In the youngest forests, N fixers grew 2.5 times faster, recruited at a similar rate and were 15 times less likely to die as non-fixers. As succession proceeded, the growth and survival disparities decreased, whereas N fixer recruitment decreased relative to non-fixers. According to our individual-based model, high survival was the dominant driver of the increase in basal area of N fixers. Our data suggest that N fixers are successful throughout secondary succession in tropical rainforests of north-east Costa Rica, and that attempts to understand this success should focus on tree survival.

Can evolutionary constraints explain the rarity of nitrogen-fixing trees in high-latitude forests?

Contents 1195 I. 1195 II. 1196 III. 1196 IV. 1200 1200 References 1200 SUMMARY: The rarity of symbiotic nitrogen (N)-fixing trees in temperate and boreal ('high-latitude') forests is curious. One explanation - the evolutionary constraints hypothesis - posits that high-latitude N-fixing trees are rare because few have evolved. Here, we consider traits necessary for high-latitude N-fixing trees. We then use recent developments in trait evolution to estimate that > 2000 and > 500 species could have evolved from low-latitude N-fixing trees and high-latitude N-fixing herbs, respectively. Evolution of N-fixing from nonfixing trees is an unlikely source of diversity. Dispersal limitation seems unlikely to limit high-latitude N-fixer diversity. The greater number of N-fixing species predicted to evolve than currently inhabit high-latitude forests suggests a greater role for ecological than evolutionary constraints.

Nitrogen fixation strategies can explain the latitudinal shift in nitrogen-fixing tree abundance.

The rarity of symbiotic nitrogen-fixing trees in higher-latitude compared to lower-latitude forests is paradoxical because higher-latitude soils are relatively N poor. Using national-scale forest inventories from the United States and Mexico, we show that the latitudinal abundance distribution of N-fixing trees (more than 10 times less abundant poleward of 35 degrees N) coincides with a latitudinal transition in symbiotic N-fixation type: rhizobial N-fixing trees (which are typically facultative, regulating fixation to meet nutritional demand) dominate equatorward of 35 degrees N, whereas actinorhizal N-fixing trees (typically obligate, maintaining fixation regardless of soil nutrition) dominate to the north. We then use theoretical and statistical models to show that a latitudinal shift in N-fixation strategy (facultative vs. obligate) near 35 degrees N can explain the observed change in N-fixing tree abundance, even if N availability is lower at higher latitudes, because facultative fixation leads to much higher landscape-scale N-fixing tree abundance than obligate fixation.

Nitrate leaching losses and the fate of 15N fertilizer in perennial intermediate wheatgrass and annual wheat - A field study.

Perennial grains, such as the intermediate wheatgrass (Thinopyrum intermedium) (IWG), may reduce negative environmental effects compared to annual grain crops. Their permanent, and generally larger, root systems are likely to retain nitrogen (N) better, decreasing harmful losses of N and improving fertilizer N use efficiency, but there have been no comprehensive N fertilizer recovery studies in IWG to date. We measured fertilizer N recovery with stable isotope tracers in crop biomass and soil, soil N mineralization and nitrification, and nitrate leaching in IWG and annual wheat in a replicated block field experiment. Nitrate leaching was drastically reduced in IWG (0.1 and 3.1 kg N ha-1 yr-1) in its third and fourth year since establishment, compared with 5.6 kg N ha-1 yr-1 in annual wheat and 41.0 kg N ha-1 yr-1 in fallow respectively. There were no differences in net N mineralization or nitrification between IWG and annual wheat, though there was generally more inorganic N in the soil profile of annual wheat. More 15N fertilizer was recovered in the straw and all depths of the roots and soils in IWG than annual wheat. However, annual wheat recovered much more 15N fertilizer in the seeds compared to IWG, which had lower grain yields. 15N-labeled fertilizer contributed little (<3 %) to nitrate-N in leachate, highlighting the role of soil microbes in regulating loss of current year fertilizer N. The large reduction in nitrate leaching demonstrates that perennial grains can reduce harmful nitrogen losses and offer a more sustainable alternative to annual grains.

Allometric relationships for eight species of 4-5 year old nitrogen-fixing and non-fixing trees.

Allometric equations are often used to estimate plant biomass allocation to different tissue types from easier-to-measure quantities. Biomass allocation, and thus allometric equations, often differs by species and sometimes varies with nutrient availability. We measured biomass components for five nitrogen-fixing tree species (Robinia pseudoacacia, Gliricidia sepium, Casuarina equisetifolia, Acacia koa, Morella faya) and three non-fixing tree species (Betula nigra, Psidium cattleianum, Dodonaea viscosa) grown in field sites in New York and Hawaii for 4-5 years and subjected to four fertilization treatments. We measured total aboveground, foliar, main stem, secondary stem, and twig biomass in all species, and belowground biomass in Robinia pseudoacacia and Betula nigra, along with basal diameter, height, and canopy dimensions. The individuals spanned a wide size range (<1-16 cm basal diameter; 0.24-8.8 m height). For each biomass component, aboveground biomass, belowground biomass, and total biomass, we determined the following four allometric equations: the most parsimonious (lowest AIC) overall, the most parsimonious without a fertilization effect, the most parsimonious without canopy dimensions, and an equation with basal diameter only. For some species, the most parsimonious overall equation included fertilization effects, but fertilization effects were inconsistent across fertilization treatments. We therefore concluded that fertilization does not clearly affect allometric relationships in these species, size classes, and growth conditions. Our best-fit allometric equations without fertilization effects had the following R2 values: 0.91-0.99 for aboveground biomass (the range is across species), 0.95 for belowground biomass, 0.80-0.96 for foliar biomass, 0.94-0.99 for main stem biomass, 0.77-0.98 for secondary stem biomass, and 0.88-0.99 for twig biomass. Our equations can be used to estimate overall biomass and biomass of tissue components for these size classes in these species, and our results indicate that soil fertility does not need to be considered when using allometric relationships for these size classes in these species.

Nitrogen fixation in the stag beetle, Ceruchus piceus (Coleoptera: Lucanidae): could insects contribute more to ecosystem nitrogen budgets than previously thought?

Nitrogen (N) is a key nutrient required by all living organisms for growth and development, but is a limiting resource for many organisms. Organisms that feed on material with low N content, such as wood, might be particularly prone to N limitation. In this study, we investigated the degree to which the xylophagous larvae of the stag beetle Ceruchus piceus (Weber) use associations with N-fixing bacteria to acquire N. We paired acetylene reduction assays by cavity ring-down absorption spectroscopy (ARACAS) with 15N2 incubations to characterize rates of N fixation within C. piceus. Not only did we detect significant N fixation activity within C. piceus larvae, but we calculated a rate that was substantially higher than most previous reports for N fixation in insects. While taking these measurements, we discovered that N fixation within C. piceus can decline rapidly in a lab setting. Consequently, our results demonstrate that previous studies, which commonly keep insects in the lab for long periods of time prior to and during measurement, may have systematically under-reported rates of N fixation in insects. This suggests that within-insect N fixation may contribute more to insect nutrition and ecosystem-scale N budgets than previously thought.